LINC complexes: connecting the nuclear lamina to the cytoskeleton
The nuclear envelope (NE) of mammalian cells is composed of two lipid bilayers, the inner and the outer nuclear membrane (INM and ONM), which are connected at nuclear pores, thus delineating the perinuclear space. The ONM is an extension of the rough endoplasmic reticulum (ER) and the INM adheres to the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins. Our work is mostly focused on Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) that consist in macromolecular assemblies that span the NE and physically connect the nuclear lamina to different components of the peripheral cytoskeleton. LINC complexes are formed by the direct interaction, within the perinuclear space, between evolutionary-conserved luminal domains of two families of integral transmembrane proteins: Sun proteins and Nesprins (Figure 1).
Sun1 and Sun2 are integral type II transmembrane proteins whose nucleoplasmic regions interact directly with both A- and B-type lamins. The luminal region of Sun1 and Sun2, which protrude into the perinuclear space, contain two coiled-coil domains and a highly conserved C-terminal region of approximately 150 amino acids called the SUN (Sad1 and UNC-84 homology) domain. This domain interacts directly with the evolutionary-conserved KASH domain (Klarsicht/Anc-1/Syne Homology) shared by Nesprin 1, 2, 3 and 4, another family of type II transmembrane proteins whose cytoplasmic region harbors multiple spectrin repeats. While different isoforms of Nesprin display a complex subcellular localization pattern, giant Nesprin 1 (>1MDa) and Nesprin 2 (>700kDa) isoforms are anchored in the ONM and extend as rod-like structures of up to 300-400 nm into the cytoplasm, where they bind F-actin through N-terminal calponin motifs. Nesprin 3 also contains a KASH domain, but its cytoplasmic region is characterized by the presence of a plectin-binding domain. Nesprin 4 also interact with Sun proteins via its KASH domain and interact with molecular motors.
The wide variety of cytoplasmic domains (spectrin repeats, actin and plectin binding domains) provided by KASH domain proteins coupled with their alternative splicing, their developmentally-regulated expression and tissue specificity underlie the wide array of physiological functions of SUN/KASH based macromolecular assemblies. Indeed, SUN and KASH domain proteins are involved in nuclear anchorage to the cytoskeleton, nuclear migration, the coupling of the centrosome to the nucleus, chromosome dynamics, cell polarization and cellular tensegrity. These physiological functions are evolutionary-conserved.
For more details, see Razafsky and Hodzic, J Cell Biol, 186(4); 461-72 (2009).
Retinogenesis is tightly coupled to precisely orchestrated nuclear dynamics events. The first one, interkinetic nuclear migration, consists in the migration of neurons precursors nuclei in phase with the cell cycle. Upon exit from the cell cycle, post-mitotic neurons then migrate towards specific laminar position. This nuclear choreography leads to the formation of a mature retina that consists in three distinct layers of nuclei. We use mouse retinas to study the function and developmental regulation of LINC complex components that underlie these dynamic nuclear events as well as primary neuronal culture to dissect the role of LINC complexes during neuronal migration that relies on nuclear translocation.
In the past decade, the nuclear envelope of mammalian cells has been the subject of intense scrutiny following the discovery that mutations of several of its components are involved in an impressive array of human diseases. To date, over 200 mutations scattered along the gene encoding A-type lamins are associated with over a dozen human pathologies collectively called laminopathies. The most common laminopathies consist in cardiomyopathy and autosomal Emery-Dreifuss muscular dystrophy. The molecular etiology of these diseases is still unknown. By virtue of their direct interaction with A- and B-type lamins, a disruptive effect of laminopathic mutations on the organization of LINC complexes is very likely. This is further reinforced by our recent observation that disruption of endogenous LINC complexes result in a loss of cellular mechanical stiffness, a phenotype that is also observed in mouse embryonic fibroblasts derived from mice lacking lamin A/C, a mouse model for Emery-Dreifuss muscular dystrophy. These results therefore suggest a mechanical etiology of muscular and cardiac laminopathies. We are currently developing transgenic mouse models as well as in vitro models to test these hypotheses.
Using biochemical approaches and molecular biology, we also attempt to identify new binding partners of Sun proteins and Nesprins. Indeed, a more comprehensive characterization of interacting networks of Sun proteins and Nesprins may lead to a better understanding of the feast of biological functions that SUN/KASH-based interactions can achieve.